Lyme borreliosis is now the most common arthropod-borne disease in the United States. Borrelia burgdorferi, the causative agent, has been isolated from humans, mammals, birds, and arthropods and is cultivable in the laboratory. Careful structural analysis of the DNA content of several early passage isolates has revealed, in addition to a 1,000 kb linear genome, a unique mixture of terminally cross-linked linear and covalently-closed, circular DNA molecules ranging in size from 2 to 50 kilobases in length. Often DNA patterns appear to differ among isolates and to vary during laboratory passage. While all of the linear species were shown to rapidly reanneal to linear duplexes after alkaline denaturation, single-stranded circular molecules measuring twice the length of the linears could be produced by treatment with methyl mercury, glyoxal and urea prior to mounting for electron microscopy. This rapid reannealing mediated by the terminal sequences of the molecule was used in a unique ion exchange column chromatography system to purify terminal, restriction fragments from any which originate internally. Purified terminal fragments could then be compared and characterized further by conventional methods. The use of rRNA sequences as chronometers of evolutionary divergence is now widespread and its advantage over classical taxonomic approaches is well documented. To date, over 500 small subunit rRNA sequences have been determined and used for this purpose. We have determined in near entirety, the 16S sequences of several borrelial species and isolates. The phylogenetic relationships among the isolates were determined through calculations of sequence similarity and Knuc values and the data presented as an unrooted phylogenetic tree. In addition, rDNA restriction patterns were determined using Southern blot analyses, and the data compared with published genomic grouping schemes. The objective of this project, therefore, is to define the genetic capacity of B. burgdorferi and other vector-borne pathogens in sufficient detail to begin the process of mapping those genes and gene products which may be important in pathogenesis.